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Quantum dots (QDs)-based single particle analysis technique enables real-time tracking of the viral infection in live cells with great sensitivity over a long period of time. The porcine reproductive and respiratory syndrome virus (PRRSV) is a small virus with the virion size of 40–60 nm which causes great economic losses to the swine industry worldwide. A clear understanding of the viral infection mechanism is essential for the development of effective antiviral strategies. In this study, we labeled the PRRSV with QDs using the streptavidin–biotin labeling system and monitored the viral infection process in live cells. Our results indicated that the labeling method had negligible effect on viral infectivity. We also observed that prior to the entry, PRRSV vibrated on the plasma membrane, and entered the cells via endosome mediated cell entry pathway. Viruses moved in a slow–fast–slow oscillatory movement pattern and finally accumulated in a perinuclear region of the cell. Our results also showed that once inside the cell, PRRSV moved along the microtubule, microfilament and vimentin cytoskeletal elements. During the transport process, virus particles also made contacts with non-muscle myosin heavy chain II-A (NMHC II-A), visualized as small spheres in cytoplasm. This study can facilitate the application of QDs in virus infection imaging, especially the smaller-sized viruses and provide some novel and important insights into PRRSV infection mechanism. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-019-00187-0) contains supplementary material, which is available to authorized users.

Virus infection in host cells is a complex process comprising a large number of dynamic molecular events. Single-virus tracking is a versatile technique to study these events. To perform this technique, viruses must be fluorescently labeled to be visible to fluorescence microscopes. The quantum dot is a kind of fluorescent tag that has many unique optical properties. It has been widely used to label proteins in single-molecule-tracking studies but rarely used to study virus infection, mainly due to the lack of an accepted labeling method. In this study, we developed a lipid-specific method to readily, mildly, specifically, and efficiently label enveloped viruses with quantum dots by recognizing viral envelope lipids with lipid-biotin conjugates and recognizing these lipid-biotin conjugates with streptavidin-quantum dot conjugates. It is not only applicable to normal viruses, but also competent to label the key protein-mutated viruses and the inactivated highly virulent viruses, providing a powerful tool for single-virus tracking., Quantum dots (QDs) possess optical properties of superbright fluorescence, excellent photostability, narrow emission spectra, and optional colors. Labeled with QDs, single molecules/viruses can be rapidly and continuously imaged for a long time, providing more detailed information than when labeled with other fluorophores. While they are widely used to label proteins in single-molecule-tracking studies, QDs have rarely been used to study virus infection, mainly due to a lack of accepted labeling strategies. Here, we report a general method to mildly and readily label enveloped viruses with QDs. Lipid-biotin conjugates were used to recognize and mark viral lipid membranes, and streptavidin-QD conjugates were used to light them up. Such a method allowed enveloped viruses to be labeled in 2 h with specificity and efficiency up to 99% and 98%, respectively. The intact morphology and the native infectivity of viruses were preserved. With the aid of this QD labeling method, we lit wild-type and mutant Japanese encephalitis viruses up, tracked their infection in living Vero cells, and found that H144A and Q258A substitutions in the envelope protein did not affect the virus intracellular trafficking. The lipid-specific QD labeling method described in this study provides a handy and practical tool to readily “see” the viruses and follow their infection, facilitating the widespread use of single-virus tracking and the uncovering of complex infection mechanisms.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes tremendous economic losses to the swine industry worldwide. miRNAs are crucial regulators of gene expression and a wide range of complex interactions of miRNAs-mRNAs is possible during virus infection. However, there is no comprehensive integrated study of miRNA and mRNA networks in MARC-145 cells after infection with PRRSV. We analyzed the differential expressions, co-relations, annotations, and putative functions of miRNA and mRNA networks in PRRSV-infected MARC-145 cells. Based on the filtering criterion, 22 differentially expressed miRNAs (DEmiRs) (15 up- and 7 downregulated) were filtered out. miRNA-mRNA interaction networks were constructed. For the 18 selected miRNAs, 390 potential target genes were predicted from the differentially expressed mRNAs (DEmRs). GO and KEGG pathway annotations predicted 34 KEGG pathways, 12 of which are known to be involved in virus infection. Real-time PCR validated the RNA-seq results. Our analysis showed that miR-27a-5p and miR-21-3p downregulate the expression of two of their potential target genes-SPARC, CLIC1, and cofilin-1, COX7A2, respectively. Further experiments proved that miR-21-3p and miR-27a-5p can promote PRRSV replication significantly. It is the first report that these two miRNAs participate in the interaction of host cells with PRRSV. Our results provide insights into the role of miRNAs in response to PRRSV infection, which will aid the research for developing novel therapies against PRRSV.

Clostera anastomosis (L.) granulovirus (CaLGV) and Clostera anachoreta granulovirus (ClanGV) are both capable of infecting each other's native host insects. Despite this, we have little information on their genetic relationship. The complete nucleotide sequence of CaLGV was determined and compared with that of the genome of ClanGV. The circular, double-stranded DNA CaLGV genome (GenBank accession no. KC179784) had a G+C content of 46.7 % and was 101,818 bp in size (331 bp larger than the ClanGV genome). Overall, the CaLGV nucleotide sequence was found to be 90 % identical to that of ClanGV. It contained a total of 123 ORFs, 119 of which had ClanGV homologues, with an identical transcription direction and ORF organization. Seventy-five of the 119 ORFs showed 90 % or greater identity to their ClanGV homologues. CaLGV contained only a single identifiable homologous region (hrs)/repeat region (similar to ClanGV hr4). The mean frequency of nucleotide substitutions in the CaLGV/ClanGV coding regions was 8.33 %. CaLGV contained four unique ORFs (CaL23, CaL39, CaL48 and CaL92). Eight ORFs found in both CaLGV and ClanGV have no homologues in other baculoviruses. Intergenic regions of CaLGV and ClanGV occupied 6.6 % and 7 % of their respective genomes. CaLGV appears closer phylogenetically to ClanGV than to any other baculoviruses.

The complete genome of Clostera anachoreta granulovirus (ClanGV) from an important pest of poplar, Clostera anachoreta (Lepidoptera: Notodontidae), was sequenced and analyzed. The circular double-stranded genome is 101,487 bp in size with a C+G content of 44.4%. It is predicted to contain 123 open reading frames (ORFs), covering 93% of the whole genome sequence. One hundred eleven ClanGV ORFs are homologues of previously reported baculovirus genes, two ORFs only exist in nucleopolyhedroviruses (NPVs), and 10 ORFs are unique to ClanGV, accounting for 3.9% of the genome in total. The average amino acid sequence identities between ClanGV and other granulovirus (GV) homologues ranged from 55.5% (PiraGV) to 43.1% (HearGV and PsunGV). ClanGV includes 16 DNA replication- and transcription-related genes, including ie-1, dnapol, helicase, lef-1, lef-2, lef-3, lef-4, lef-5, lef-6, lef-8, lef-9, lef-10, lef-11, 39K, p47 and vlf-1. Seventeen conserved structural genes and two types of apoptosis suppression proteins (P35 and IAP) have also been identified. To date, ClanGV is the second identified GV that contains the p35 gene, which is present in eight NPVs and ChocGV. Genes of chitinase and cathepsin, which are involved in the liquefaction of the host, were found in the ClanGV genome, consistent with the typical insecticidal properties of ClanGV. In the ClanGV genome, there are four homologous repeat regions, each one of which contains only 2-3 direct repeats. Phylogenetic analysis, based on the 29 core baculovirus genes, indicates that ClanGV is closely related to PhopGV, AdorGV, CpGV, CrleGV, PiraGV and ChocGV, and this agrees with the results of homology analysis and gene parity plot analysis.

In the present study, the genomic sequence characteristics of HN-JY40 strains of the hepatitis E virus (HEV) isolated from pigs in Henan Province, China, were analyzed and the evolutionary relationship between HN-JY40 and other sequenced strains examined. The whole genome of HN-JY40 was sequenced and analyzed by reverse transcription-polymerase chain reaction, 3' rapid amplification of cDNA ends (3' RACE) and 5' RACE. Bioinformatic analyses were carried out with Megalign, Expasy, clustal x, and MEGA 4 software. The genome of HN-JY40 was 7 223 bp in size upon removal of polyA sequences. Sizes were 9 bp and 69 bp at 5' and 3' noncoding regions, respectively. The genome of HN-JY40 was predicted to contain three open reading frames (ORFs): ORF1 (5 124 bp) encoding 1 707 amino acids; ORF2 (2 025 bp) encoding 674 amino acids; ORF3 (345 bp) encoding 114 amino acids. Phylogenetic-tree analyses indicated that HN-JY40 is a typical type-IV virus that belongs to a new subgenotype of HEV genotype 4. We sequenced and analyzed the whole genome of HN-JY40. This strategy elicited the genomic characteristics of the HEV isolated from pigs in Henan Province as well as the evolutionary relationships between HN- JY40 and other HEV isolates from pigs. We revealed that the ORF1 of HN-JY40 (153-432 nt) and human HK 104-2004 had high similarity, which offers molecular evidence for uncovering the interspecies transmission of the HEV.

To date, proteomic studies have been performed on occlusion-derived viruses (ODVs) from five members of the family Baculoviridae, genus Alphabaculovirus, but only a single member of the genus Betabaculovirus (Pieris rapae granulovirus). In this study, LC-MS/MS was used to analyse the ODV proteins of Clostera anachoreta granulovirus (ClanGV), another member of the genus Betabaculovirus. The results indicated that 73 proteins, including the products of 27 baculovirus core genes, were present in ClanGV ODVs. This is the largest number of ODV proteins identified in baculoviruses to date. To the best of our knowledge, 24 of these proteins were newly identified as ODV-associated proteins. Twelve of the proteins were shared by all seven of the other baculoviruses that have been analysed by proteomic techniques, including P49, PIF-2, ODV-EC43, P74, P6.9, P33, VP39, ODV-EC27, VP91, GP41, VLF-1 and VP1054. ClanGV shared between 20 and 36 ODV proteins with each of the other six baculoviruses that have been analysed by proteomics. Ten proteins were identified only as ODV components of ClanGV and PrGV: Clan22, Clan27, Clan69, Clan83, Clan84, Clan90, Clan116, Clan94, FGF-3 and ME53, the first seven of which were encoded by betabaculovirus-specific genes. These findings may provide novel insights into baculovirus structure as well as reveal similarities and differences between alphabaculoviruses and betabaculoviruses.

The complete nucleotide sequence of Agrotis segetum granulovirus Shanghai strain (AgseGV-L1) was determined and compared with that of AgseGV Xinjiang strain (AgseGV-XJ). The circular genome of AgseGV-L1 is 131,442 bp and has a G+C content of 37.27 %. It includes 149 ORFs, 24 of which are unique to AgseGV (AgseGV-L1 and AgseGV-XJ [GenBank accession no. NC_005839]). The average level of amino acid sequence identity between AgseGV-L1 and other granulovirus (GV) homologues (except AgseGV-XJ) ranged from 44.3 % (Adoxophyes orana granulovirus [AdorGV]) to 49 % (Cydia pomonella granulovirus [CpGV]). The AgseGV-L1 genome is 99 % identical to that of AgseGV-XJ. They have 196 differences, including 172 substitutions, 21 deletions and 3 insertions. Two homologous regions (hrs) were detected in two intergenic spaces, which share low identity and both lack a palindromic core. A p6.9 gene was found in this genome, which shared 38 %-59 % amino acid identity with those of other baculoviruses. No differences were found in the hr and p6.9 sequences of AgseGV-L1 and AgseGV-XJ. Ie-1 is a known immediate-early gene, but AgseGV-L1 ie-1 has no recognizable promoter element. By BLASTP analysis, one bro gene homologue of NPVs was detected (Agse148). Phylogenetic analysis based on the 29 core baculovirus genes indicated that AgseGV-L1 is closely related to AgseGV-XJ, Helicoverpa armigera granulovirus (HearGV), Helicoverpa armigera granulovirus (XecnGV), Pseudaletia unipuncta granulovirus (PsunGV), Spodoptera litura granulovirus (SpliGV) and Plutella xylostella granulovirus (PlxyGV).

The BamHI-C restriction fragment of ClanGV genome was cloned and sequenced. Sequence analysis indicated that this region contains five important baculovirus homologous genes (chitinase, cathepsin, gp37, p49 and odv-e18) and one ORF unique to ClanGV genome. The genes, located within this restriction fragment, were compared with homologues in other baculoviruses. Comparison results indicated that ClanGV, Cydia pomonella GV and Pieris rapae GV have similar arrangement and orientation of the homologous genes. The ORFs related to these five genes were analyzed.

A novel electrode array was developed based on the XNA on Gold(TM) microarray platform. The platform combines self-assembling monolayers, thick film patterning and streptavidin based immobilization to provide a robust, versatile platform capable of analysing virtually any biomolecule including nucleic acids, proteins, carbohydrates and lipids. Electrochemical analysis of the self-assembling monolayer/streptavidin (SAMS) XNA on Gold(TM) coating revealed that the ferrocene redox current for the SAMS modified electrode was greater than that with a bare Gold(TM) electrode. The electrochemical reaction of K4Fe(CN)(6) was inhibited by the SAMS coating, but was reactivated upon addition of ferrocene. These results indicate that ferrocene is involved as a mediator in the electron transfer of K4Fe(CN)(6) to the SAMS modified electrode. Addition of DNA to the SAMS resulted in only a minor change in the electrochemical signal, indicating that XNA on Gold(TM) can be used for electrochemical based bioanalysis. After cycling a SAMS electrode 50 times, no signs of deterioration were detected showing that coating has excellent stability. In addition to the biosensing applications, the scheme provides a non-invasive method for accessing the quality of the SAMS coatings which is of industrial interest. These studies show that the XNA on Gold(TM) microarray platform can be used for electrochemical studies, thus providing an additional alternative for developing multianalyte biosensors as well as expanding the range of detection methods available for microarray analysis. (C) 2004 Elsevier B.V. All rights reserved. (Less)